US20200061610A1 - Device, system and method relative to the preconcentration of analytes - Google Patents

Device, system and method relative to the preconcentration of analytes Download PDF

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Publication number
US20200061610A1
US20200061610A1 US16/348,475 US201716348475A US2020061610A1 US 20200061610 A1 US20200061610 A1 US 20200061610A1 US 201716348475 A US201716348475 A US 201716348475A US 2020061610 A1 US2020061610 A1 US 2020061610A1
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observation
channels
channel
nanochannel
microchannel
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Francois-Damien Delapierre
Antoine PALLANDRE
Stephane Guilet
Anne-Marie Haghiri-Gosnet
Edmond Cambril
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Centre National de la Recherche Scientifique CNRS
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/413—Concentration cells using liquid electrolytes measuring currents or voltages in voltaic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44752—Controlling the zeta potential, e.g. by wall coatings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0896—Nanoscaled

Definitions

  • the invention concerns the field of devices, systems, and processes for analyzing and/or discriminating and/or sorting particles or molecules present in a solution (more generally referred to as analytes, which may be charged or uncharged, contained in an electrolyte).
  • a solution more generally referred to as analytes, which may be charged or uncharged, contained in an electrolyte.
  • the invention also makes it possible to carry out chemical and biochemical reactions by mixing very locally two groups of molecules present in very small quantities.
  • the invention proposes a solution in the field of microfluidics exploiting the competition of electrophoretic mobility, and advantageously hydrodynamic (additional pressure, Poiseuille flow) and electroosmotic mobility, and more particularly electropreconcentation.
  • FIG. 1 shows a glass microchannel filled with an ionic solution.
  • the pH is higher than about 2, the walls are negatively charged. Consequently, cations (illustrated by “+”) in the bottom electrolyte accumulate near the walls. The cations closest to them cannot move, but the next layers of cations move to a cathode K when an electric field is applied, entraining all the liquid.
  • anions illustrated by “ ⁇ ”
  • cations everywhere else in the liquid also migrate to the anode A and the cathode K, respectively.
  • electrophoretic flow This is called electrophoretic flow, which depends on the charge/mass ratio of the analyte.
  • the first observations of the phenomenon of ionic preconcentration around a nanoslit were made by Pu et al. in 2004 [Pu-2004] (references are detailed at the end of the description).
  • the device that allowed these observations represented schematically in FIG. 2 , consists of two 100 ⁇ m-deep, U-shaped microchannels M 1 , M 2 connected by eight 60 nm-deep nanochannels N 0 at the curvature of the U. Each nanochannel forms a nanometric restriction that disrupts anion transfer, which generates the preconcentration phenomenon.
  • Preconcentration is thus notably a function of the nature and/or geometry of the restriction and the biophysical characteristics of the analyte, such as its mobility.
  • FIGS. 3 a and 3 b show the results before and after electropreconcentration.
  • the assembly is filled with a solution containing fluorescein or rhodamine 6G diluted in sodium tetraborate solution ( FIG. 3 a ).
  • concentration polarization FIG. 3 b
  • An enrichment factor of 100 and a depletion (scarcity) factor of 500 were observed.
  • a simple model based on charge transfer balances and considerations of a double electric layer within the nanochannel was proposed by the authors.
  • the device studied this time consists of two microchannels M 1 , M 2 connected by a so-called horizontal-slit nanochannel Nh. This is called a Micro/Nano/Micro (MNM) device.
  • the channels are formed in a plate P extending in a plane XY and are covered by a cover plate C, typically a thin material.
  • Nanochannels extend along the axis X included in the plane XY.
  • a horizontal-slit nanochannel corresponds to a portion of channel whose depth, i.e. the dimension along the axis Z that is orthogonal to the plane XY, is less than the depth of the two microchannels on either side (see FIG. 4 ).
  • the simulated solutions consist of fluorescein diluted in KCl solutions of variable ionic strengths.
  • An accumulation of analytes in a particular location is referred to as a concentration spot or focal point.
  • this flow profile moves without deformation from top to bottom and this focal point changes position. If the concentration spot is against the nanoslit, it is referred to as “stacking”; if it is far, “focusing”. This stacking and focusing can be anodic or cathodic.
  • AS Anodic Stacking
  • AF Anodic Focusing
  • CF Cathodic Focusing
  • CS Cathodic Stacking
  • a similar horizontal-slit nanochannel Nh device was used by Lou ⁇ r et al. [Louer-2013].
  • the device integrates a 100 ⁇ m-long and 150 nm-deep nanochannel into a 1 ⁇ m-deep microchannel (see FIG. 4 ).
  • the document WO2010034908 also describes a horizontal nanochannel (see FIG. 10 b for example).
  • Sung et al. [Sung-2012] have developed a device based on a channel between two reservoirs, blocked by a porous membrane (Nafion membrane).
  • the channel has a constant width of 50 ⁇ m.
  • a variant of the device consists in using different reservoir lengths to observe variations in preconcentration.
  • the document WO2010052387 presents a device in which the disturbance is generated by a particular polarizable coating.
  • chromatography techniques exclusion, ion exchange, affinity
  • gel electrophoresis Western Blot
  • capillary techniques capillary techniques.
  • the separated proteins are analyzed with different means (dichroism, spectrophotometry, NMR, mass spectrometry, X-ray). Immunological methods can also be used (ELISA for example).
  • concentration spots generated are not always sufficiently accurate, which prevents the characterization of molecules or particles.
  • the architecture of the device is not adapted to observe the spots, particularly in a desire to analyze, discriminate or sort particles or molecules.
  • the horizontal nanoslits have a width that does not favor the development of compact and efficient devices.
  • the invention proposes a device, system and associated process to solve each of the above-mentioned problems.
  • the invention concerns a device for the detection/selective preconcentration of charged analytes contained in an electrolyte, comprising:
  • observation channels each extending in a longitudinal direction in a plane
  • each observation channel being notably formed by:
  • a nanochannel comprising a first end and a second end, and having a length
  • nanochannels have an area section smaller than the section of the at least one microchannel, wherein, for at least two nanochannels, one dimension among their respective length or their respective section is different.
  • the device can include the following characteristics, taken alone or in technically possible combination:
  • the device comprises two microchannels, the first microchannel extending from the first supply conduit to the first end of the nanochannel, the second microchannel extending from the second supply conduit to the second end of the nanochannel,
  • the device comprising at least three observation channels, wherein each nanochannel has at least one of said dimensions different from the nanochannel of the other observation channels,
  • the section of a channel defines a width, parallel to the plane and orthogonal to the longitudinal axis, and a depth, orthogonal to the plane and to longitudinal axis, wherein the nanochannels have a width comprised between 50 and 500 nm, and the microchannels have a width comprised between 1 and 20 ⁇ m, preferably between 1 and 10 ⁇ m,
  • the section of a channel defines a width, parallel to the plane and orthogonal to the longitudinal axis, and a depth, orthogonal to the plane and longitudinal axis, wherein at least two nanochannels, preferably all, have different widths two to two.
  • the section of a channel defines a width, parallel to the plane and orthogonal to the longitudinal axis, and a depth, orthogonal to the plane and to the longitudinal axis, wherein the nanochannels have equal lengths.
  • the section of a channel defines a width, parallel to the plane and orthogonal to the longitudinal axis, and a depth, orthogonal to the plane and to the longitudinal axis, wherein at least two nanochannels, preferably all the nanochannels, have different lengths in pairs,
  • the microchannels have different lengths
  • the section of a channel defines a width, parallel to the plane and orthogonal to the longitudinal axis, and a depth, orthogonal to the plane and to the longitudinal axis, wherein at least one microchannel has a width comprised between 4 and 6 ⁇ m, preferably 5 ⁇ m, and/or a depth between 0.8 and 1.2 ⁇ m,
  • observation channels extend in a straight line and parallel to each other
  • the device comprises a plate defining the plane, wherein the observation channels are etched on one side of said plate, and comprising a cover plate for covering the observation channels,
  • the two microchannels of said observation channel have different widths and/or lengths
  • the invention concerns a device for the detection/selective preconcentration or of charged analytes contained in an electrolyte, comprising:
  • observation channel connected to each other by the observation channel and configured to convey the electrolyte the observation channel, the observation channel comprising:
  • the device can comprise the following characteristics, taken alone or in technically possible combination:
  • the device comprises two microchannels, the first microchannel extending from the first supply conduit to the first end of the nanochannel, the second microchannel extending from the second supply conduit to the second end of the nanochannel,
  • the depth of the nanochannel is equal to the depth of the microchannels, the depth of a channel being the dimension of said channel orthogonally to the plane,
  • the observation channel is etched on one side of said plate, the device further comprising a cover plate to cover at least said observation channel,
  • the nanochannel has a width comprised between 50 and 500 nm, and the at least one microchannel has a width comprised between 1 and 20 ⁇ m, preferably between 1 and 10 ⁇ m,
  • the device comprises at least two observation channels wherein one observation channel is referred to as primary, another observation channel is referred to as secondary, the two channels being positioned in series between the supply conduits,
  • the device includes a plurality of observation channels positioned in parallel between the two supply conduits,
  • the plate comprises a layer of silicon or glass, a layer of silicon dioxide, in which the observation channel is etched,
  • the two microchannels of an observation channel have different widths and/or lengths.
  • the invention concerns a system comprising one of the two devices as described above, and further comprising at least two ports, for fluid communication with the supply channels, and comprising electrodes configured to be placed in the ports and a controllable electrical voltage source capable of generating a potential difference within the observation channels via the electrodes.
  • the system can further comprise a controllable hydrodynamic pressure generator, associated with at least one of the ports and capable of generating a pressure gradient within the observation channels.
  • a controllable hydrodynamic pressure generator associated with at least one of the ports and capable of generating a pressure gradient within the observation channels.
  • the system further comprises advantageously an imaging device configured to acquire video/images from the observation channels.
  • the invention concerns a process for the detection/selective preconcentration of charged analytes contained in an electrolyte using one of the two devices as described above or using a system as described above, comprising the following steps:
  • E 2 application of a voltage at the ends of the observation channels for a specified period of time to cause an electropreconcentration phenomenon and allow analyte concentration spots to appear in the at least one microchannel.
  • Step E 2 may further comprise the application of a pressure gradient within the observation channels for a specified period of time.
  • the process comprises an additional step E 3 of measuring at least one of the following characteristics on each of the microchannels of the different observation channels (for at least two observation channels), for each spot:
  • the process can comprise an additional step E 4 of comparing the characteristics obtained in step E 3 with a database to obtain information about the analyte.
  • the invention concerns a process for manufacturing one of the two devices as defined above, comprising the following steps:
  • a preparation step F 1 comprising the following steps:
  • an etching step F 2 comprising the following steps:
  • the device can comprise the following characteristics, taken alone or in technically possible combination:
  • the observation channel etching step F 22 consists in etching in a single step the at least one microchannel and the nanochannel, the microchannel ( 120 ) having a width greater than the width of the nanochannel ( 110 ),
  • the microchannels have a width comprised between 1 and 20 ⁇ m, preferably between 1 and 10 ⁇ m, and the nanochannels have a width comprised between 50 nm and 500 nm,
  • the silicon dioxide layer etching step F 2 is carried out by a jet orthogonal to said layer
  • observation channels are spaced in pairs at a distance comprised between 15 and 100 ⁇ m, preferably between 30 and 50 ⁇ m,
  • the process comprises the additional steps F 4 of:
  • the process comprises a step F 5 of mounting a thin glass cover plate, to close the channels, preferably by thermal bonding without intermediate resist.
  • the cover is mounted using an intermediate bonding layer, hydrogen silsequioxane.
  • FIG. 1 already presented, illustrates the general functioning of flow competition
  • FIGS. 2, 3 a , 3 b , 4 already presented, illustrate devices of the prior art
  • FIG. 5 already presented, illustrates the modes of preconcentration
  • FIGS. 6 and 7 illustrate a system in which a device in accordance with several embodiments of the invention is positioned
  • FIG. 8 illustrates a top view of one embodiment of a so-called vertical-slit device in accordance with the invention
  • FIGS. 9 a , 9 b illustrate two cross-sectional views (longitudinal and orthogonal to each other) of a so-called vertical-slit device in accordance with the invention
  • FIGS. 10, 10 b illustrate the same two cross-sectional views of a so-called horizontal-slit device of the prior art
  • FIG. 11 illustrates one possible embodiment of the device with two observation channels in series, in accordance with the invention
  • FIGS. 12, 13, 14 a to 14 c illustrate a device conforming to a so-called barcode embodiment, in accordance with the invention
  • FIGS. 15 a to 15 c illustrate the preconcentration results in each of the channels of a so-called barcode device, in accordance with the invention
  • FIGS. 16 a to 16 f represent photographs from which the results of FIGS. 15 a to 15 c were derived
  • FIG. 17 illustrates the steps of an analyte detection/analysis/identification process, in accordance with the invention
  • FIG. 18 illustrates the difference in the positioning of the concentration spots in the different channels of a so-called barcode device, in accordance with the invention
  • FIG. 19 illustrates the underlying physical principle for discriminating between two analytes using a barcode device, in accordance with the invention
  • FIG. 20 similar to FIG. 18 , illustrates the difference in the positioning of concentration spots in the different channels of a so-called barcode device in accordance with the invention, for two different analytes contained in the same electrolyte,
  • FIG. 21 illustrates different layers for manufacturing a device in accordance with the invention
  • FIG. 22 illustrates the different steps of manufacturing a device in accordance with the invention.
  • the devices 10 concerned here are devices for the detection/selective preconcentration of charged or uncharged analytes contained in an electrolyte. These devices also allow chemical and biochemical reactions to be carried out very locally by mixing several groups of molecules, generally present in small quantities.
  • These devices are of the Micro-Nano-Micro (MNM) type, i.e., in the broadest sense, comprising a succession of at least two channels of varying sizes. These devices 10 operate by applying an electrical voltage to its terminals, optionally coupled to a hydrodynamic pressure.
  • MNM Micro-Nano-Micro
  • the device 10 is configured to receive electrolytes containing analytes, hereinafter referred to as solution.
  • FIGS. 6 and 7 illustrate a general system in which the device is positioned.
  • the device 10 comprises an observation channel 100 , type MNM, within which electropreconcentration phenomena occur.
  • the observation channel 100 extends in a longitudinal direction X. It can be created in different modes, which will be described below.
  • the observation channel 100 is filled with solution.
  • the device 10 has at least two openings 102 , 104 , configured to allow a potential difference to be applied in the observation channel 100 using electrodes 30 , 32 . To that end, each opening 102 , 104 is in fluid communication with one of the ends of the observation channel 100 .
  • a system comprises the device 10 , a seal 20 typically made of polydimethylsiloxane (PDMS), a block 22 typically made of polymethyl methacrylate (PMMA), assembled one on top of the other and held in place by clamping means 24 such as screws.
  • the PMMA block comprises two ports 22 a , 22 b opposite the openings 102 , 104 .
  • the ports 22 a , 22 b are configured to be filled with solution (for example using a syringe) and are of sufficient size to each accommodate an electrode 30 , 32 (for example a platinum electrode).
  • the purpose of the seal 20 positioned between the block 22 and the device 10 , is to seal the connection, since the solution level must reach the electrodes which are not in the same plane as the ports 102 , 104 .
  • More than two ports 22 a , 22 b and two openings 102 , 104 can be provided, to be able to multiply the number of electrodes forming anode and cathode. This allows for faster filling and easier cleaning.
  • the electrodes 30 , 32 are connected to a controllable voltage generator 34 ( FIG. 7 ) that can typically deliver a potential difference ranging from a few dozen volts to several hundred volts.
  • the voltages it must be able to generate will be specified below according to the embodiments of the device 10 .
  • the generator covered a range from ⁇ 110V to +110V.
  • a certain number of electronic devices 36 can be coupled to the generator to allow better control or measurement of the applied voltages or electrical characteristics of the system (impedance, etc.).
  • One of the two ports 22 a , 22 b thus corresponds to the anode A and the other to the cathode K.
  • the ports 22 a , 22 b are configured to receive a controllable pressure generator 40 .
  • This generator 40 allows a hydrodynamic pressure difference to be applied along the observation channel 100 .
  • the pressure controller 40 can be connected to flexible tubes 42 , 44 that can be inserted into or encircle the ports 22 a , 22 b may be suitable.
  • pressure values are determined together with the electrical voltage values. Nevertheless, pressures below 1 bar are suitable for moving the spots and making them readable.
  • An imaging device 50 is provided in the system, to acquire video/images from the observation channel 100 .
  • the device 50 generally comprises a microscope 52 coupled to a camera 54 .
  • filters can be used and the lighting can be adapted.
  • a mercury lamp with filters can be used to excite and observe fluorescent analytes (for wavelengths in the visible therefore).
  • the imaging device 50 must be able to acquire measurements of low fluorescence intensity to monitor analyte concentration.
  • the microscope 52 is usually coupled with a shutter (Uniblitz VCMD1, for example) to limit photobleaching.
  • One method of rapid identification is to optically read the fluorescence.
  • the control of the voltage generator 34 and, if need be, the pressure generator 40 is carried out by a calculation unit 60 which can also directly retrieve the images acquired by the imaging device 50 .
  • the observation channel 100 is defined as having a length Lo, along the longitudinal axis X and sections orthogonal to said axis X.
  • the device 10 consists of three parts in particular: a reservoir part where the solution arrives, an observation zone, connected to the reservoir part, where electropreconcentration occurs, and a disturbance zone, connected to two observation zones on either side of the disturbance zone.
  • the observation zone and the disturbance zone form the observation channel 100 .
  • the observation zone generally has a section whose minimum dimension is of the order of one micrometer, while the disturbance zone has a section whose minimum dimension is of the order of one hundred nanometers.
  • this generic name MNM cannot be restrictive because it can encompass observation zones whose minimum dimension is of the order of hundred nanometers.
  • FIG. 8 shows a general embodiment of a device 10 .
  • the device comprises a plate P defining a plane XY.
  • the longitudinal direction X is parallel to this plane XY.
  • One embodiment of the manufacturing of the plate will be detailed below. It is generally made of glass or silica, but not only these.
  • the observation channel 100 is formed in the plate.
  • the observation channel 100 comprises a nanochannel 110 with a first end 110 a and a second end 110 b .
  • the nanochannel 110 forms the disturbance zone.
  • the observation channel 100 comprises a first microchannel 120 a connected to the first end 110 a of the nanochannel 110 and a second microchannel 120 b connected to the second end 110 b of the nanocal 110 .
  • the two microchannels 120 a , 120 b are generically referenced 120 .
  • the three channels 110 , 120 a , 120 b are therefore arranged in series and preferably in a straight arrangement. A total length Lo of the observation channel 100 can then be defined.
  • Microchannel refers to a channel, i.e. a slit, made in the plate.
  • Micro- and nano- generally and without limitation refer to a minimum dimension of the section, which is either of the order of one micrometer or of the order of one hundred nanometers. More precisely, nano- and micro- mean that a dimension is of the order of a hundred nanometers in one channel and that the same dimension is of the micrometric order in another channel.
  • a first supply conduit 130 a and a second 130 b are connected to the first observation channel 110 a and the second 110 b , respectively.
  • the supply conduits form the above-mentioned reservoir part. Their function is to provide a sufficient volume of solution to allow the proper use of the device 10 .
  • the supply conduits each comprise two separate reservoirs, which meet through an intermediate channel 132 at the end of the first microchannel 120 a and the second microchannel 120 b , respectively.
  • the dimensions of the sections of the supply conduits are generally greater than 100 ⁇ m to provide a sufficiently large amount of solution for preconcentration to occur in the microchannels 120 .
  • the supply channels 130 a , 130 b thus form two symmetrical dihedra transversely to the longitudinal axis X, each dihedron being symmetrical with respect to the longitudinal axis X.
  • the openings 102 , 104 are formed opposite the supply conduits 130 a , 130 b in general at a zone 134 of greater dimension than the width of the other parts of the supply conduits 130 a , 130 b .
  • there are two zones 134 (associated with two openings 102 , 102 and 104 , 104 and two ports 22 a , 22 a , and 22 b , 22 b ), symmetrically placed along the longitudinal axis X.
  • the two symmetrical zones 134 can be located between 6 and 10 mm apart. In the case of the previous dihedron, the zones 134 are at the ends of said dihedra.
  • a width I is defined along the axis Y, i.e. the dimension of the channel parallel to the plane XY and orthogonal to the longitudinal axis X
  • a depth P is defined along the axis Z, which is the dimension of the channel orthogonally to the longitudinal axis X.
  • the axes X and Y form a direct angle in the plane XY, and the trihedron XYZ is direct.
  • the sections of the channels can be rectangular but this involves greater cleaning constraints (particularly because of stagnant zones). Sections with rounded angles provide more stability to the concentration region and improve reproducibility (because it avoids the release effects of stagnant zones). In particular, these so-called rounded sections form transition zones between the microchannels 120 and the nanochannel 110 .
  • a cover plate C covers the channels (observation channel 100 and supply channel 130 ) to isolate the assembly. As previously mentioned, openings 102 , 104 are provided in the plate or cover to allow fluid communication.
  • the section S 110 of the nanochannel 110 has a smaller area than the section S 120 of the microchannel 120 . Quantities will be explained below.
  • FIGS. 9 a and 9 b represent one embodiment of a so-called “vertical-slit” device.
  • the vertical-slit observation channel is defined by the dimensions of the widths of the microchannels 120 and of the nanochannel 110 .
  • the width I 120 of the microchannels 120 is comprised between 1 and 20 ⁇ m, preferably 1 and 10 ⁇ m.
  • the width of the nanochannel 110 is comprised between 50 and 500 nm, preferably between 100 and 400 nm.
  • a restriction is then observed in the plane XY, more precisely along the axis Y, which generates a disturbance.
  • the depth P along the axis Z is constant throughout the observation channel 100 , i.e. the depth Pm of the microchannels 120 is the same as the depth P 110 of the nanochannel 110 .
  • a depth of around 1 ⁇ m is appropriate. Nevertheless, a depth between 100 and 500 nm may also be appropriate, so that the two dimensions of the nanochannel section are of the order of 100 nanometers.
  • the microchannel 120 has a micrometric dimension and a nanometric dimension and the nanochannel 110 has two nanometric dimensions.
  • the width I 120 and the length of the first microchannel 120 a are equal to the width and length of the second microchannel 120 b , respectively.
  • microchannels I 120 may be expressly provided to have different widths of microchannels I 120 . This provides an additional discrimination factor, notably for complex electrolytes, i.e. electrolytes containing a plurality of different analytes with different sizes and chemical groups.
  • the length of the first microchannel 120 a may be different from the length of the second microchannel 120 b.
  • the vertical-slit device makes it possible to concentrate analytes by a factor of 1000.
  • FIGS. 10 a and 10 b represent one embodiment of a so-called “horizontal-slit” device 100 . These devices were presented in the introduction and are already known.
  • the width of the microchannel I 120 is equal to the width along the axis Y of the nanochannel I 110 .
  • the depth along the axis Z of the nanochannel P 110 is less than the depth of the microchannel P 120 .
  • the depth of the nanochannel P 110 is comprised between 75 and 350 nm and the depth of the microchannel P 120 is comprised between 2 and 3 ⁇ m, for example at about 2.5 ⁇ m.
  • the preconcentration phenomenon is visible in a direction orthogonal to the nanometric dimension, i.e. the width of the nanochannel 110 , easily accessible for the imaging device 50 .
  • the total surface area of the device can be reduced (see in particular the barcode device below), which allows more results to be obtained for the equivalent device size. This allows more results to be obtained for a single experiment, avoiding repeatability contingencies and saving time and material: the ideal parameters can be found in the same operation.
  • microchannel 120 a or 120 b In a particular embodiment, which applies to any vertical- or horizontal-slit observation channel 100 , only one microchannel 120 a or 120 b is provided. Consequently, the nanochannel 110 is connected on the other side directly to the supply channel 130 .
  • the arrangement, in series, is thus as follows: supply channel 130 a , microchannel 120 a , nanochannel 110 , supply channel 130 b.
  • This embodiment although not symmetrical, can be advantageously used with the application of hydrostatic pressure that allows the concentration spot to be moved.
  • microchannels 120 a , 120 b The present description has been made for two microchannels 120 a , 120 b but applies to the device with only one microchannel.
  • the observation channel 100 is called primary and the device comprises at least one other observation channel called secondary, located between the primary observation channel 100 and one of the supply channels 120 a , 120 b , so that the primary 100 and secondary 100 ′ channels are arranged in series.
  • a buffer zone ZT is provided between the two observation channels 100 , 100 ′.
  • More than two observation channels 100 , 100 ′ can thus be provided in series.
  • the primary 100 ′ and secondary 100 ′ observation channels may have identical dimensions for reasons of symmetry. Alternatively, having different primary 100 and secondary 100 ′ observation channels makes it possible to observe more different behaviors for the analytes present in the electrolyte.
  • Hydrodynamic pressure can also be applied at the zone ZT, replacing or complementing the other application locations described above.
  • the device must then be adapted, particularly in terms of opening, ports and sealing.
  • the “vertical-slit” observation channel is particularly suitable for a so-called “barcode” device 10 . Indeed, the latter is easy to manufacture and generates localized spots Sp.
  • a horizontal-slit device can also be used.
  • the barcode device 10 comprises a plurality of observation channels 100 a , 100 b , . . . each extending in a longitudinal direction X in a plane XY.
  • the observation channels 100 a , 100 b are parallel to each other.
  • one dimension among their respective length or their respective section S 110 is different. In other words, all the nanochannels 110 do not have the same length or the same section S 110 .
  • nanochannel 110 verifying a given length/section pair, or the number of redundancies, i.e. observation channels with the same characteristics, is limited.
  • the barcode device 100 comprises two vertical-slit observation channels 100 a , 100 b , the following configurations may be available:
  • each microchannel 120 a , 120 b of each of the observation channels 100 preconcentration is likely to be observed.
  • the length Lo is the same for all the observation channels 100 in order to simplify the manufacture of the device 100 . Nevertheless, if necessary, a condition may be the equal length of the microchannels 120 (especially when the nanochannels 110 have different lengths).
  • the observation channels are spaced in pairs at a distance comprised between 15 and 100 ⁇ m, preferably between 30 and 50 ⁇ m. The shorter this distance, the more the device can be compact or integrate a large number of observation channels 100 .
  • the supply channels 130 a , 130 b play an important role as buffer and reservoir. Indeed, it is important that each observation channel 100 can be considered as independent and that there is no mixing between the microchannels 120 a , 120 b . In other words, this means that the volume of solution present in the supply channels is considered infinite from a theoretical point of view. A value of 100 ⁇ m for width 1130 is suitable. A depth equal to the depth of the microchannels 120 is appropriate.
  • the preconcentration phenomenon depends on several factors, notably including the disturbance generated in the channel. Here it is the nanochannel.
  • the preconcentration spots Sp are modified.
  • the position, motion, width, intensity of each preconcentration spot is information that can help identify the observed analyte
  • the plurality of observation channels 100 a , 100 b , 100 c allows a multiplicity of information to be acquired in a single experiment.
  • analysis of the behaviors of the spots Sp on the different observation channels 100 allows the analyte to be identified.
  • FIGS. 15 a , 15 b , 15 c illustrate the behavior of an analyte in the different observation channels as a function of time, for a variant in accordance with FIG. 14 a .
  • Observations include:
  • FIGS. 16 a to 16 f show a preconcentration created with a 10 ⁇ M sodium fluorescein solution in a 10 ⁇ M KCl solution.
  • This device has three nanoslits of different widths of 100, 200 and 300 nm (from top to bottom in the photos). A voltage of 20 V was applied and a pressure of 0.2 bar in the opposite direction to the electroosmotic flow. The observation time is 50 s.
  • the preconcentration for these parameters is carried out well within the intermediate zone, i.e. in the intermediate microchannels 120 a and 120 b , provided for that purpose.
  • the parameter matrix of the analyte can be formed.
  • controllable pressure generator 40 allows the preconcentration conditions to be modified.
  • the barcode device 100 Using the barcode device 100 , more precisely using a system comprising such a device, it is possible to implement a process for the detection or identification, or a process for the selective preconcentration, of charged analytes contained in an electrolyte ( FIG. 17 ).
  • a first step E 1 the supply channels 130 , the micronals 120 and the nanochannels 110 are filled with solution. This filling can be done by injection via a syringe.
  • electrodes 30 , 32 are positioned in the openings 22 a , 22 b of the system and a voltage is applied using the controllable voltage source 34 .
  • the voltage is applied for a period of time specified in the protocol. Under the effect of the voltage, preconcentration phenomena can occur, which generates the appearance of concentration sports of analytes in the microchannels 120 .
  • a hydrodynamic pressure can be applied on one of the ports 22 a , 22 b using the controllable pressure generator 40 .
  • a measurement and analysis step E 3 at least one of the following data is acquired for at least two observation channels 100 a , 100 b (see FIG. 18 ):
  • Each additional parameter refines the matrix and is therefore able to reduce the set of molecules that can correspond to it.
  • measurements are acquired for each observation channel, the physical characteristics (length, width, depth) of which are known.
  • a step E 4 the data obtained and/or processed are compared with a database.
  • the data can be arranged in a matrix for example.
  • the analyte By comparing with a database, for example differences between data, the analyte can be characterized or identified, so that at least one piece of information about the analyte can be obtained. Furthermore, depending on whether hydrodynamic pressure is applied, the mapping may change. A mapping without pressure and a mapping with different pressures can thus be obtained.
  • the database is generated by calibration on several model solutions containing known analytes.
  • the solution does not contain a single type of analyte but several.
  • the barcode device 10 makes it possible to select and discriminate these analytes.
  • FIG. 19 illustrates the physical basis. Indeed, a molecule or protein with high mobility under electric field tends to concentrate on the cathode side K, while a molecule or protein with low mobility under field tends to preconcentrate on the anode side A. Mention may be made of fluorescein and bovine albumin serum, respectively.
  • the controllable pressure generator 40 can then play an important role in making the device 100 effective on analytes.
  • the plate P of the device advantageously comprises a silicon or glass layer and a silicon dioxide SiO 2 layer, in which the observation channel is etched.
  • the SiO 2 layer is purer than silicon or glass and its etching is easier to control than glass.
  • the cover plate C usually in the form of a thin material, which covers the channels, can be made of glass.
  • the barcode device particularly the vertical-slit device, makes it possible to analyze solutions containing trace or ultra-trace biomolecules, such as threat and biohazard pathogens.
  • the analytes can be particles, nanoparticles, viruses, proteins, etc.
  • the fields concerned are scientific research, chemical and bacteriological risk management, pollutant monitoring, drug quality control, detection of disease markers in biological fluids.
  • a vertical-slit device 100 requires trenches of varying widths to be etched into a material. This trench must have a controlled width and a relatively large depth to allow for a reasonable flow rate.
  • a depth of 1 ⁇ m is suitable, but not limiting. In particular, any depth between 0.8 ⁇ m and 5 ⁇ m is suitable, and preferably between 1.8 ⁇ m and 1.2 ⁇ m.
  • Substrate refers to the assembly of the different layers that are processed to manufacture the device 100 (see FIG. 21 ). This term is generic and applies to assemblies at any stage.
  • the described process therefore applies for a device comprising a single vertical slit or a plurality of vertical slits in series, or for a “barcode” device comprising a plurality of vertical slits in parallel and optionally in series.
  • the manufacturing process distinguishes between several main steps ( FIG. 22 ):
  • a silicon dioxide SiO 2 layer referenced 210 , is deposited.
  • the thickness of this layer 210 depends in particular on the depth desired for the observation channel 100 . In this case, a thickness of 1 ⁇ m is suitable.
  • the layer is deposited by plasma-enhanced chemical vapor deposition (PECVD).
  • PECVD plasma-enhanced chemical vapor deposition
  • the thickness of the glass or silicon plate has no direct impact on the preconcentration. On the other hand, the thickness must be chosen to allow the observation of preconcentration spots (focal length of the lens, absorption of the glass). This plate is also thermalized during subsequent etching, which can change the etching parameters.
  • Silicon dioxide is softer and purer than silicon and glass. It is this layer 210 , therefore, that will be etched.
  • a thin layer of germanium 220 is deposited on the SiO 2 .
  • a thick layer of aluminum 230 is deposited. Its thickness is comprised between 150 and 250 nm, and is preferably 200 nm.
  • the germanium intermediate layer only reduces the roughness of the aluminum layer.
  • a fourth step F 14 the aluminum is covered with a layer of positive electron-sensitive resist 240 , type “ZEP520A”.
  • This layer 240 has a thickness comprised between 400 and 600 nm, and is preferably 500 nm.
  • This resist is for example deposited by spin coating on the surface at 2000 rpm for 30 s (500 rpm/s acceleration), then annealed 30 min at 160° C.
  • a fifth step F 15 the electron-sensitive resist is exposed in electron lithography, i.e. electron beam lithography, in order to draw the patterns that will be etched.
  • the resist is developed for 1 min 15 s in a developer (for example ZED-N50).
  • the assembly can be rinsed with isopropanol.
  • the open patterns in the resist expose the aluminum.
  • the substrate is then ready for the etching step F 2 .
  • a first step F 21 the aluminum is etched with chlorine plasma by inductively coupled plasma reactive ion etching (ICP-RIE) to reveal the pattern on the SiO 2 .
  • ICP-RIE inductively coupled plasma reactive ion etching
  • a second step F 22 the SiO 2 is then etched with fluorine (SF6)/oxygen/argon plasma.
  • SF6 fluorine
  • SF6 the energy injected into the plasma
  • bias the ions to bombard the surface
  • the proportions of the gases the energy injected into the plasma
  • the ionic bombardment is activated only part of the time (30-50% over 500 ms), so that the plasma without bombardment, more isotropic, smooths the walls of the etched patterns, and that the charges deposited on the surface by the bombardment are removed. The latter indeed led to a more pronounced etching in the vicinity of the walls.
  • the characteristics used were as follows: ⁇ 13 min, 5 mTorr, SF6 (60 sccm), O2 (3 sccm), Ar (7 sccm), ICP: 300 W, bias: 30 W.
  • the etching step consists in etching all the channels in a single step.
  • the dimensions of the micro- and nanocals have been presented above.
  • the etching depth is ideally the same in the micro- and nanochannel.
  • the etching is stopped before reaching the end of the silicon dioxide layer. Ideally, the etching is configured to stop at the end of the layer.
  • the pattern is then finished. The assembly is then ready for the cleaning step F 3 .
  • a first step F 31 the remaining resist is removed by washing, typically with acetone and trichloroethylene.
  • a second step F 32 the unetched metal, protected by the resist, is removed, typically by dissolution with soda or piranha mixture (H 2 SO 4 (50%)/H 2 O 2 (50%)).
  • a resist type AZ5214.
  • the purpose of this layer is to prevent debris from getting trapped in the nanoslit.
  • the layers of the substrate are drilled to generate the openings 102 , 104 which allow the injection of solution into the channels.
  • the drilling is done transversely.
  • the openings 102 , 104 must therefore open opposite the supply channels.
  • the ports are opened in the substrate with a microbead blaster or a drill.
  • a metal guide i.e. a steel block pierced with holes, is used. These holes are arranged so that they can be aligned with all the ports of the device.
  • the device is bonded to this block, the holes in front of the parts of the pattern to de drilled.
  • a microbead jet is projected from the other side of the guide and erodes the glass or silica at the openings of the metal until it is completely pierced.
  • the substrate is cleaned with trichloethylene, acetone, water, isopropanol and piranha mixture.
  • the substrate is then ready to be covered.
  • a fifth step F 5 the substrate, on the side of the etched channels, is closed with a cover C, typically made of a thin glass.
  • the bonding of this lamella can be done in various ways: thermal bonding (bringing the substrate and the cover into contact, under vacuum, under high pressure and temperature).
  • a preferential embodiment uses a resist, hydrogen silsesquioxane (HSQ), as an intermediate bonding layer using the process described in the document FR 1054183.
  • HSQ hydrogen silsesquioxane
  • the device 10 is then ready to be mounted in the system presented above, allowing the injection of liquid and the application of an electrical voltage.
  • dry plasma etching is preferred to wet etching.
  • the ions constituting the plasma are accelerated with an electric field and bombard the surface to be etched perpendicular thereto. Etching is therefore preferentially done in this direction. Dry etching is therefore likely to present a certain anisotropy.
  • chemical reactions occur independently of direction and the substrate is isotropically etched.
  • etching is done in only two steps (etching of the metal and etching of the silicon dioxide) that can be performed successively on the same machine.
  • the manufacturing process involves electron nanolithography and etching.

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